International Journal of Molecular Sciences Article A Novel Multiprotein Bridging Factor 1-Like Protein Induces Cyst Wall Protein Gene Expression and Cyst Differentiation in Giardia lamblia Shao-Wei Huang 1,†, Zi-Qi Lin 1,†, Szu-Yu Tung 1, Li-Hsin Su 1, Chun-Che Ho 1, Gilbert Aaron Lee 2 and Chin-Hung Sun 1,* 1 Department of Tropical Medicine and Parasitology, College of Medicine, National Taiwan University, Taipei 100, Taiwan; [email protected] (S.-W.H.); [email protected] (Z.-Q.L.); [email protected] (S.-Y.T.); [email protected] (L.-H.S.); [email protected] (C.-C.H.) 2 Department of Medical Research, Taipei Medical University Hospital, Taipei 110, Taiwan; [email protected] * Correspondence: [email protected]; Tel.: +886-2-23123456-88262; Fax: +886-2-23915294 † These authors contributed equally to this work. Abstract: The capacity to synthesize a protective cyst wall is critical for infectivity of Giardia lamblia. It is of interest to know the mechanism of coordinated synthesis of three cyst wall proteins (CWPs) during encystation, a differentiation process. Multiprotein bridging factor 1 (MBF1) gene family is a group of transcription coactivators that bridge various transcription factors. They are involved in cell growth and differentiation in yeast and animals, or in stress response in fungi and plants. We asked whether Giardia has MBF1-like genes and whether their products influence gene expression. BLAST searches of the Giardia genome database identified one gene encoding a putative MBF1 protein with a helix-turn-helix domain. We found that it can specifically bind to the AT-rich initiator promoters of the Citation: Huang, S.-W.; Lin, Z.-Q.; encystation-induced cwp1-3 and myb2 genes. MBF1 localized to cell nuclei and cytoplasm with higher Tung, S.-Y.; Su, L.-H.; Ho, C.-C.; Lee, expression during encystation. In addition, overexpression of MBF1 induced cwp1-3 and myb2 gene G.A.; Sun, C.-H. A Novel Multiprotein Bridging Factor 1-Like expression and cyst generation. Mutation of the helixes in the helix-turn-helix domain reduced cwp1- Protein Induces Cyst Wall Protein 3 and myb2 gene expression and cyst generation. Chromatin immunoprecipitation assays confirmed Gene Expression and Cyst the binding of MBF1 to the promoters with its binding sites in vivo. We also found that MBF1 can Differentiation in Giardia lamblia. Int. interact with E2F1, Pax2, WRKY, and Myb2 transcription factors that coordinately up-regulate the J. Mol. Sci. 2021, 22, 1370. https:// cwp genes during encystation. Using a CRISPR/Cas9 system for targeted disruption of mbf1 gene, doi.org/10.3390/ijms22031370 we found a downregulation of cwp1-3 and myb2 genes and decrease of cyst generation. Our results suggest that MBF1 is functionally conserved and positively regulates Giardia cyst differentiation. Academic Editor: Amelia Casamassimi Received: 4 January 2021 Keywords: cyst; MBF1; transcription; Giardia; differentiation; DNA binding protein; parasite; tran- Accepted: 26 January 2021 scription regulation Published: 29 January 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. Giardia lamblia parasitizes the human small intestine to cause diarrheal disease world- wide [1,2]. Poor hygiene and water resource contamination can result in the transmission of giardiasis [1,3,4]. Chronic diarrhea due to giardiasis may lead to malnutrition and growth failure in children [5–7]. Recently, Giardia infection has been recognized as a cause of higher risk of irritable bowel syndrome with persisting abdominal symptoms [8,9]. Copyright: © 2021 by the authors. Giardia Licensee MDPI, Basel, Switzerland. As a single-cell protozoan, is a valuable model for understanding the evolution This article is an open access article of cell differentiation [1]. The presence of fewer basic components for biological pathway distributed under the terms and suggest that Giardia exhibits unusual mechanisms as compared with other eukaryotes [10]. conditions of the Creative Commons Like many other protozoa that persist in a dormant state, Giardia differentiates from a Attribution (CC BY) license (https:// pathogenic trophozoite into a resistant walled cyst, which is a tactic for resistance of hypo- creativecommons.org/licenses/by/ tonic lysis in fresh water and survival in gastric acid during transmission [1,3]. Proteins 4.0/). and polysaccharides are the major components of the cyst wall, whose encystation-specific Int. J. Mol. Sci. 2021, 22, 1370. https://doi.org/10.3390/ijms22031370 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 1370 2 of 24 synthesis is key to encystation [11–13]. During encystation, three cyst wall proteins (CWPs) are newly synthesized to form cyst wall [14–16]. It is of interest to identify factors involved in up-regulation of cwp genes. Several transcription factors are induced in cyst differentia- tion, including Myb2 (Myb1-like protein in the Giardia genome database), GARP1, ARID1, WRKY, E2F1, Pax1, and Pax2 and participate in this process [17–24]. Bacteriophage Cro and CI (λ) repressors contains typical helix-turn-helix (HTH) do- mains of about 50–60 residues [25,26]. The basic core of HTH domain contains three α-helices (H1, H2 and H3) separated by two short turns [25,26]. H2 and H3 are critical for DNA interaction [25,26]. H3 is the recognition helix that is needed for DNA bind- ing and recognizes specific sequence along the DNA major groove [25,26]. H2 can help nonspecifically stabilize the complex [25,26]. H1 has a slight role in DNA binding. The Cro/CI-type HTH domain are present in many transcription regulators of prokaryotes and eukaryotes [25,26], including multiprotein bridging factor 1 (MBF1). MBF1 proteins contain a highly conserved HTH domain with four α-helixes [27,28]. MBF1 may have DNA binding activity because HTH domain is a typical DNA binding domain [27,28]. MBF1 has not been found in bacteria [27]. The archaea MBF1 proteins are quite different from the eukaryotic ones as they have an extra Zn ribbon motif [27]. MBF1 was named because it functioned as a transcriptional coactivator by bridging regulatory transcription factors and general transcription factors in higher eukaryotes [29]. MBF1 proteins have been found in yeast and human and play important roles in regulating cell proliferation and differentiation [29–32]. MBF1 is a co-activator that bridges GCN4, a transcriptional activator of the bZIP family that regulates amino acid biosynthesis in yeast [32]. MBF1 is also named as endothelial differentiation-related factor 1 (EDF1) as it plays a role in differentiation of epithelial cells in human [33]. MBF1 interacts with a sequence specific DNA binding factor of steroid receptor family, fushi tarazu factor 1 (FTZ-F1), to induce transcription of fushi tarazu gene which encodes a product working in Drosophila early embryo segmentation [34]. MBF1 interacts with a general transcription factor, TATA-binding protein, and a gene-specific transcription factor, FTZ-F1, for FTZ gene transactivation by stabilizing the protein-DNA interactions in development of silkworm Bombyx mori [35]. In addition, MBF1 gene was up-regulated by a virus that causes cell death of lobsters, suggesting that MBF1 is stress-related [36,37]. MBF1 functions as a positive modulator of AP-1 for its DNA binding by preventing an oxidative modification of AP-1 during Drosophila oxidative stress [38]. MBF1 may be involved in stress tolerance and in ethylene signal transduction pathway for seed germination in Arabidopsis [39]. MBF1 also plays a role in stress response in fungi [40]. In another aspect, MBF1 proteins play roles in translation process in archaea and yeast [41,42]. Little is known about MBF1 in protozoan parasites. To date, only Cryptosporidium MBF1 has been characterized [43]. It is a transcription co-activator working with TATA- binding protein [43]. Because of the critical roles of MBF1 proteins in cell proliferation and cell differentiation of many eukaryotes, we asked whether Giardia has MBF1 family proteins and whether they function in inducing Giardia differentiation into dormant cysts. We searched the Giardia genome database and identified one MBF1 homolog (MBF1). We found that MBF1 was up-regulated during encystation and it possessed DNA-binding activity to AT-rich initiator sequences. We also found that MBF1 can interact with important encystation-induced E2F1, Pax2, WRKY, and Myb2 transcription factors. Using overex- pression and mutation analysis, and a CRISPR/Cas9 system [44] for targeted disruption of the mbf1 gene, we found that MBF1 can up-regulate cwp1-3 and myb2 genes and induce cyst generation. Our results suggest that MBF1 may be an important transcription factor promoting Giardia differentiation. 2. Results 2.1. Analysis of the mbf1 Gene An MBF1 homolog (GenBank accession number XP_001704109.1, open reading frame 2732) was identified using a BLAST search of the G. lamblia genome database using the Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 25 of the mbf1 gene, we found that MBF1 can up-regulate cwp1-3 and myb2 genes and in- duce cyst generation. Our results suggest that MBF1 may be an important transcription factor promoting Giardia differentiation. 2. Results Int. J. Mol. Sci. 2021 22 , , 1370 2.1. Analysis of the mbf1 Gene 3 of 24 An MBF1 homolog (GenBank accession number XP_001704109.1, open reading frame 2732) was identified using a BLAST search of the G. lamblia genome database using the sequencesequence ofof humanhuman MBF1MBF1 (GenBank(GenBank accessionaccession numbernumber NP_003783.1)NP_003783.1) asas aa queryquery [[10,45].10,45]. TheThe deduced deducedGiardia GiardiaMBF1 MBF1 protein protein is is composed composed of of 107 107 amino amino acids acids with with a a pI pI of of 10.22 10.22 and and aa molecularmolecular weightweight ofof 11.97 kDa. Like Like human human MBF1, MBF1, the the GiardiaGiardia MBF1MBF1 has has a aputative putative 4- 4-helixhelix HTH HTH DNA-binding DNA-binding domain domain near near C-terminus C-terminus as as predicted predicted by by Pfam Pfam (Figures (Figure S1S1 and and Figure1A) [29,46].1A) [29 ,46].